This invention relates to the isolation and purification of lipid cell-wall components originating from bacteria assigned to the genera Mycobacterium, Corynebacterium, Nocardia or Rhodococcus, of which the most ubiquitous and the most important from a human health point of view is the genus Mycobacterium.
The genus Mycobacterium comprises a large number of both saprophytic and pathogenic species. The best known members of the genus, M. tuberculosis and M. leprae, are the causative agents of tuberculosis and leprosy, respectively, both among the most serious diseases occurring in man.
Tuberculosis: Current status
Tuberculosis is considered to be the major communicable disease throughout most of the world. Despite great advances in medical science and a range of effective drugs, which for some time created the impression that the disease had been conquered, and despite organised international efforts, tuberculosis remains a world health problem of staggering proportions: approximately one third of the world's population is infected with M. tuberculosis (Fauci, 1995), more than 8 million new cases world-wide and more than 3 million deaths in the year 1990 alone were reported (Snider, 1994). Predictions made by the World Health Organisation indicate that by the year 2000 the annual figures will grow to 10,2 million new cases and 3,5 million deaths, with Asia and sub-Saharan Africa being the most affected continents (De Cock et al., 1992; Dolin, Raviglione and Kochi, 1994; Raviglione, Snider and Kochi, 1995; Wilkinson and de Cock, 1996). According to the recently released "WHO Report on the Tuberculosis Epidemic, 1995" the figures anticipated for the next decade are even more alarming: 300 million new infections and 30 million deaths (Holler, 1995). In effect, tuberculosis was declared in 1993 by WHO to be a global public health emergency (Bloomfield, 1995; Wilkinson and de Cock, 1996).
The major reasons for this dramatic comeback and the unabated spread of tuberculosis can be identified as:
1) Insufficient protection offered by the world-wide vaccination programme based on the use of BCG*) PA1 2) Problems associated with the detection of tuberculosis PA1 3) Problems associated with treatment of tuberculosis and the occurrence of multi-drug resistant strains of M. tuberculosis PA1 4) Interaction with HIV infection PA1 5) Socio-economic aspects PA1 i) Variations between BCG vaccines, which could be caused by strains variation or by differences between manufacturing processes; PA1 ii) Differences in pathogenesis of M. tuberculosis; PA1 iii) Differences in the exposure to the environmental mycobacteria--the environmental mycobacteria may act antagonistically or synergistically with BCG; PA1 iv) Genetic differences between population groups subjected to vaccination with BCG; PA1 v) Differences in nutrition and exposure to sunlight between various population groups; PA1 vi) Differences between designs of various studies; PA1 vii) Inadequacies of the criteria used for the evaluation of protective action of vaccination with BCG. PA1 i) corynomycolic acids (C.sub.28 -C.sub.40 acyl chain length) PA1 ii) nocardomycolic acids (C.sub.40 -C.sub.60 acyl chain length) PA1 iii) mycobacterial mycolic acids (C.sub.60 -C.sub.90 acyl chain length). PA1 dissolving the extracted mixture or synthetic mixture in a bi-phasic solvent to form a solution; PA1 allowing the solution to separate to form an upper phase and a lower phase; PA1 subjecting the phases to countercurrent distribution (CCD) separation comprising a required number of cycles to separate the microbial cell-wall component or analog or derivative thereof in the upper phase or the lower phase; and PA1 removing the separated microbial cell-wall component or derivative or analog thereof from the upper or lower phase.
*) BCG: (Bacillus of Calmette and Guerin) Calmette and Guerin attenuated a strain of M. bovis by passing it 231 times over a period of 13 years through a medium containing glycerine and oxbile.
1. Insufficient Protection Offered By The World-Wide Vaccination Programme Based On BCG
Attempts to prevent the spread of tuberculosis by inducing resistance to the infection with M. tuberculosis were initiated at the beginning of this century, using vaccination with BCG. On the basis of a number of controlled studies it was established that the protective efficacy obtained in vaccination with BCG varied between 0 to 80% (Snider, 1994; Hershfield, 1995) and, on the basis of the analysis of the published literature, the BCG vaccination was found to be roughly 50% effective (Colditz et al., 1994; O'Brien, 1995). A number of hypotheses/explanations for this unsatisfactory situation have been put forward (Fine, 1994). The most important are:
2. Problems Associated With The Detection Of Tuberculosis
The accurate and timely detection of tuberculosis and related mycobacterial diseases is one of the important requirements for the development of a more successful global strategy to combat these diseases.
Traditional laboratory detection methods have major disadvantages of either not being capable of distinguishing between live and dead bacilli (the quick and simple Ziehl-Neelsen staining) or, if these methods confirm the presence of the live bacilli (direct cultivation), a number of weeks is required before the laboratory tests are completed. This in turn, may delay the commencement of treatment and may lead to further spread of the disease.
Although recently developed molecular approaches to the diagnosis of tuberculosis (Godfrey-Faussett, 1994; Richeldi, Barnini and Saltini, 1995; Bloomfield, 1995; Vlaspolder, Singer and Roggeveen, 1995) resulted in the introduction of these rapid and sensitive detection tools by advanced laboratories in the developed countries, they are expensive and require specially trained personnel. For these reasons they are not suitable for screening/detection of tuberculosis in resource-poor, TB-endemic regions, already overburdened with the cost of controlling the disease (O'Brien, 1995; Voelker, 1995).
A similar situation exists in the field of rapid drug sensitivity testing (Schaberg et al., 1995; Pretorius et al., 1996) and rapid culturing of Mycobacterium (Bloomfield, 1995). The significant advances in these areas cannot be utilised, for financial reasons, in the countries most affected by the tuberculosis pandemic.
3. Problems Associated With Treatment Of Tuberculosis And The Occurrence Of Multi-Drug Resistant Strains Of M. tuberculosis
The development of effective chemotherapy for tuberculosis made the treatment of infected persons possible, thus preventing the full development of the disease. Although the anti-tuberculosis drugs with proven bacterial action (rifampicin, isoniazid and pyrazinamide) as well as the ones with bacteriostatic or resistance-preventing properties (streptomycin sulphate, ethambutol and thiacetazone) are available (Weil, 1994), worldwide success in combating the disease has not been achieved so far due to two main factors: patients' non-compliance with the prescribed regimen and financial limitations existing in developing countries.
The impact of the isoniazid preventative therapy on the control of tuberculosis in developing countries is uncertain. This approach, although widely practiced in North America, has two major disadvantages. Firstly, it may have to given for the duration of the TB sufferers' life and, secondly, its cost, i.e. US$18 per patient per 6-month course may be prohibitive, particularly in the most affected areas where an amount of US$4 per patient per year is available for total health care (O'Brien, 1995).
The interrupted and/or uncompleted treatment, apart from the detrimental effects on the individual concerned, has contributed to the emergence and spread of multi-drug resistant strains of M. tuberculosis, which further complicate the overall situation (Beyers et al., 1996). The recent WHO estimates indicate that 50 million people worldwide may already be carrying strains of M. tuberculosis resistant to one or more of the most common anti-TB drugs. It was established already in 1991, that one third of all TB patients in New York were resistant to at least one drug and almost 20% were resistant to rifampicin and isoniazid combined (Henderson 1995).
4. Interaction With HIV Infection
The close association documented between tuberculosis and HIV infection as well as the frequently concomitant presence of both these diseases add gravity to the situation (Torres et al., 1990; De Cock, 1994; Cantwell and Binkin, 1994; Murray, 1994; Antonucci et al.. 1995; Mofeson et al., 1995; Davies, Wilkinson and Colvin, 1996; Wilkinson and Moore, 1996). The number of people who develop tuberculosis in Asia because of the parallel infection with the HIV is expected to increase seven-fold this decade, according to the Public Health Reports (1995).
The emergence of multiple-drug resistance among the strains of M. tuberculosis and other atypical mycobacteria has introduced an additional dimension to this problem (Blumberg, Miller and Koornhof, 1994; Morse, 1994; Yew and Chau, 1995).
5. Socio-Economic Aspects
Other reasons contributing to a further spread of the disease, such as unemployment, overcrowding, general lowering of economic conditions, alcoholism and erosion of the public health infrastructure have been recently reviewed by Darbyshire (1995), Fauci (1995), Law et al., (1995) and Mangtani et al., (1995).
The increased influx of immigrants from the endemic areas to countries where tuberculosis has been well under control, e.g. United States of America, creates additional problems in containing the spread of tuberculosis (Huebner and Castro, 1995). The incidence of tuberculosis among immigrants to the USA is reported to be 12 times higher than that observed among the native-born population (Ballew and Becker, 1995).
The trends discussed above place an overwhelming pressure on the improvement of existing approaches to case-management of TB patients both in endemic areas and developing countries and on the development of new drugs capable of preventing and/or combating tuberculosis (Cole, 1995; Voelker, 1995). Although cautious optimism can be detected among the researchers involved in both fields (Mwinga, 1995; Grosset, 1995), currently prevailing economic restrictions, even in the developed countries, place serious limitations on funding required to develop new anti-tuberculosis drugs, the cost of which is estimated at US$150 million for each new compound (Grosset, 1995).
Immunological Prospects For Prevention And Treatment
In view of the seriousness of the problems listed above, the limitations associated with the existing methods of combating tuberculosis and the high costs of developing new forms of chemoprophylaxis and chemotherapy, immunological approaches could provide a relevant and realistic alternative to finding an effective and economically affordable solution to the control and treatment of tuberculosis and associated conditions.
Results obtained from a number of trials using treatment with killed cells of M. vaccae (Stanford and Grange, 1994) suggest that this saprophytic microorganism may find application in immunotherapy of tuberculosis either as a single agent (Bahr et al., 1990a; Bahr et al., 1990b; Stanford et al., 1990a) or in conjunction with chemotherapy (Stanford et al., 1990b; Prior et al., 1995; Onybebujoh et al., 1995).
The modulation of inflammatory processes attributed to the use of thalidomide has recently been utilised in combating tuberculosis. The beneficial effects of thalidomide on the clinical manifestation of tuberculosis reported by Cole (1995) provide grounds for considering the immunomodulatory properties of this drug in the treatment of the disease. These effects of thalidomide are attributed to its potent inhibitory action on tumour necrotic factor, a cytokine involved in the inflammatory processes accompanying tuberculosis.
The advantages stemming from these two approaches may be extended to treatment of drug-resistant forms of tuberculosis. Encouraging data have been reported in this respect from a number of trials undertaken in Iran (Etemadi, Farid and Stanford, 1992) and in Kuwait, Rumania, Vietnam and India (Stanford and Grange, 1993).